ABSTRACT Bacterial keratitis is a vision threatening disease and contributes significantly to world blindness. Given the severity of the disease, immediate, broad spectrum antibiotics such as fourth generation fluoroquinolones are crucial for successful treatment. However, rising resistance coupled with limited industry-driven drug development highlights the need for novel ophthalmic antimicrobial therapeutics. The central goals of this proposal are to address the antimicrobial void through the development of novel ophthalmic antibiotics. Preliminary data has demonstrated the promise of rifampicin+polymyxin B/trimethoprim (PT), a combination of FDA-approved antibiotics that displays broad-spectrum, synergistic activity with reduced propensity to develop resistance. A central goal of this proposal is to advance rifampicin+PT with in vivo efficacy studies in order to move this combination toward the FDA?s 505 (B)2 fast track approval process. However given the quickening pace of antibiotic resistance, novel classes of drugs must also be concurrently developed. In that regard, a high throughput chemical library screen for novel, first-in-class agents with activity against Pseudomonas aeruginosa and Staphylococcus aureus, the most common causes of keratitis, has been initiated and initial lead compounds identified. Importantly, the screening paradigm is designed to identify compounds with anti-biofilm activity, a growth state that is notoriously challenging to eradicate in human disease including ocular infections, and has not previously been the target of high throughput drug discovery screens. As part of hit-to-lead prioritization, biologic and physiochemical studies will be performed, a CRISPR-interference essential gene knock down strain set will be created in order to facilitate the identification of cellular targets of lead compounds, and a mouse model of keratitis will be established. These latter two tool systems directly enable the proposed research but also have wide-ranging utility with respect to understanding essential genes/pathways in keratitis pathogenesis. This research program provides advanced training in drug discovery that will simultaneously define the clinical promise of rifampicin+PT, identify novel agents that kill biofilm-associated cells and develop high yield tools for antimicrobial or pathogenesis studies. In addition, this proposal details a career development plan for Dr. Rachel Wozniak, MD, PhD in order to facilitate her transition to independence. Dedicated mentorship, course work and specialized seminars will contribute to an expanding knowledge of experimental design and advanced research techniques. She has obtained full institutional and departmental support for these endeavors had has demonstrated the necessary skill set, work ethic and intellectual curiosity necessary for success.